RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the "stringent" response and regulator of cellular metabolism. The classical "long" RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture

No-go decay (NGD) targets mRNAs with stalls in translation elongation for endonucleolytic cleavage in a process involving the Dom34 and Hbs1 proteins. The crystal structure of a Schizosaccharomyces pombe Dom34-Hbs1 complex reveals an overall shape similar to that of eRF1-eRF3-GTP and EF-Tu-tRNA-GDPNP. Similarly to eRF1 and GTP binding to eRF3, Dom34 and GTP bind to Hbs1 with strong cooperativity,

Tetracycline resistance protein Tet(O), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational GTPase with significant similarity in both sequence and structure to the elongation factor EF-G. Here, we present an atomic model of the Tet(O)-bound 70S ribosome based on our cryo-electron microscopic reconstruction at 9.6-Å resolution. This atomic model allow

Translational GTPases (trGTPases) are involved in all four stages of protein biosynthesis: initiation, elongation, termination and ribosome recycling. The trGTPases Initiation Factor 2 (IF2) and Elongation Factor G (EF-G) respectively orchestrate initiation complex formation and translocation of the peptidyl-tRNA:mRNA complex through the bacterial ribosome. The ribosome regulates the GTPase cycle

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that

Translation termination in eukaryotes is governed by the concerted action of eRF1 and eRF3 factors. eRF1 recognizes the stop codon in the A site of the ribosome and promotes nascent peptide chain release, and the GTPase eRF3 facilitates this peptide release via its interaction with eRF1. In addition to its role in termination, eRF3 is involved in normal and nonsense-mediated mRNA decay through its

Background: SelB is the dedicated elongation factor for delivery of selenocysteinyl-tRNA to the ribosome. In archaea, only a subset of methanogens utilizes selenocysteine and encodes archaeal SelB (aSelB). A SelB-like (aSelBL) homolog has previously been identified in an archaeon that does not encode selenosysteine, and has been proposed to be a pyrrolysyl-tRNA-specific elongation factor (EF-Pyl).

Background. Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment an

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational chang

Translocation of the tRNA·mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4°C, 20°C, and 37°C. The binding affinity of EF-G is higher to GDP than to GTP at 4°C, but lower at 37°C. The binding enthalpy and entropy chan

During the stringent response, Escherichia coli enzyme RelA produces the ppGpp alarmone, which in turn regulates transcription, translation and replication. We show that ppGpp dramatically increases the turnover rate of its own ribosome-dependent synthesis by RelA, resulting in direct positive regulation of an enzyme by its product. Positive allosteric regulation therefore constitutes a new mechan

RelE/RelB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes

In a recent issue of Molecular Cell, Studer and Joseph (2006) clarified the molecular basis of the "standby model" and demonstrated how two-step binding of mRNAs to the small ribosomal subunit depends on initiator tRNA and initiation factors, with far-reaching implications for posttranscriptional regulation of gene expression.

This theoretical work covers structural and biochemical aspects of nucleotide binding and GDP/GTP exchange of GTP hydrolases belonging to the family of small GTPases. Current models of GDP/GTP exchange regulation are often based on two specific assumptions. The first is that the conformation of a GTPase is switched by the exchange of the bound nucleotide from GDP to GTP or vice versa. The second i

Bacterial protein synthesis involves four protein factors that belong to the GTPase family: IF2, EF-G, EF-Tu, and RF3. Their role in translation has attracted considerable interest over the recent decades. Cryoelectron microscopy has made it possible to monitor the dynamics of the ribosome upon binding of the translation factors, and biochemical findings have associated the structural data with fu

In eukaryotes, termination of mRNA translation is triggered by the essential polypeptide chain release factors eRF1, recognizing all three stop codons, and eRF3, a member of the GTPase superfamily with a role that has remained opaque. We have studied the kinetic and thermodynamic parameters of the interactions between eRF3 and GTP, GDP and the non-hydrolysable GTP analogue GDPNP in the presence (K

Background. During the translation of mRNA into polypeptide, elongation factor G (EF-G) catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recen

After peptide release by a class-1 release factor, the ribosomal subunits must be recycled back to initiation. We have demonstrated that the distance between a strong Shine-Dalgarno (SD) sequence and a codon in the P site is crucial for the binding stability of the deacylated tRNA in the P site of the posttermination ribosome and the in-frame maintenance of its mRNA. We show that the elongation fa

RNA polymerases differ functionally and structurally in the initiation phase of transcription, when polymerization of 8-12 nucleotides occurs, from the later phases of transcription. Here we argue that the ribosome also might have different properties when translating the first codons in open reading frames, as compared with the later phases of translation.